Cardiovascular disease is the leading cause of death in the United States, and costs millions of dollars per year. Atherosclerosis is the leading cause of death in the developed world and nearly the leading cause in the developing world. Atherosclerosis is a disorder in which the coronary arteries become clogged by the build up of plaque along the interior walls of the arteries, leading to decreased blood flow which can in turn cause hypertension, ischemias, strokes and, potentially, death.
Atherosclerosis has been shown to occur in sites of complex hemodynamic behavior. Surgical intervention is often employed to treat it, and may include insertion of a balloon catheter to clean out the plaque, and insertion of a stent within the vessel to enable it to remain open, or may include multiple bypasses of the clogged vessels. Bypass surgery involves the removal of a section of vein from the patient's lower leg, and its transplant into the appropriate cardiac blood vessels so that blood flows through the transplanted vein and thus bypasses the clogged vessels.
A major problem associated with bypass surgery is the patency of the vessels to be used in the bypass. The bypass vessels are prone to failure, which may occur within a short period of time after bypass surgery, or after a period of several years. Hemodynamic forces have been implicated as a major factor contributing to the failure of the bypass vessels.
Hemodynamic forces (i.e., forces due to blood flow) are known to influence blood vessel structure and pathology. The vascular cells lining all blood vessels are endothelial cells, which are important sensors and transducers of the two major hemodynamic forces to which they are exposed: wail shear stress (“WSS”), which is the fluid frictional force per unit of surface area, and hoop stress, which is driven by the circumferential strain (“CS”) of pressure changes. Wall shear stress acts along the blood vessel's longitudinal axis. Circumferential strain is associated with the deformation of the elastic artery wail (i.e., changes in the diameter of the vessel) in response to the pulse of vascular pressure. Wave reflections in the circulation and the inertial effects of blood flow cause a phase difference, the stress phase angle (“SPA”), between CS and WSS. The SPA varies significantly throughout the circulation, and is most negative in disease prone locations, such as the outer walls of a blood vessel bifurcation. Hemodynamic forces have been shown to dramatically alter endothelial cell function and phenotype (i.e., high shear stress [low SPA] is associated with an atheroprotective gene expression profile, and a low shear stress [large SPA] is associated with an atherogenic gene expression profile). There is thus a great need to study vascular biology in a complete, integrative, and controlled hemodynamic environment.
Despite the significance of hemodynamic WSS and CS acting on the vessel wall, especially at regions of the circulation with a high risk of localization of cardiovascular diseases, detailed knowledge of the combined influence of the time varying patterns of WSS and CS on endothelial cell biological response has remained technologically unfeasible.
Laboratory studies of vascular fluid mechanics have demonstrated that wall shear stress (WSS) and circumferential strain (CS) are out of phase temporally, and that there is a systematic variation of the stress phase angle (SPA) throughout the circulation. This variation is highly out-of-phase in the large arteries, where arterial disease generally occurs, while in the smaller vessels and veins where disease is rare, this variation is generally in-phase.
Where an artery bifurcates, SPA varies with the local spatial position within that bifurcation, the more out-of-phase environment being localized on the outer wail of the bifurcation where atherosclerosis occurs. SPA was found to be more out-of-phase in the coronary arteries than at any other location in the circulation.
Prior technology has focused on the individual effects of WSS or CS, individually, on endothelial cells. Berthiaume and Frangos described a device that simulates wall shear stress using a rod and plate system that is similar to the cone and plate system used in viscometers. Chang described a parallel flow chamber used to simulate steady flow. Carosi et al. and Sumpio et al. describe devices to simulate cyclic strain that consists of a flexible membrane that is stretched by a motor or a vacuum suction system.
Qiu and Tarbell described a device to simulate pressure and flow in tubes, but the device did not permit using a wide range of phase angles (SPAs), and was technically difficult to use. Limitations, however, of the Qiu and Tarbell system included having the maximum attainable phase angle being 100 degrees, the amplitude and phase of the flow and pressure are coupled, and the system utilized large quantities of fluid. The present invention, by its selection of tubing and vessel diameters, in contrast, employs approximately one fifth the volume of fluid as that system. Seliktar et al., in an in vitro study, verified that simulation of the hemodynamic environment is critical to vessel patency and function.
The patent literature described several systems for examining the effects of strain, or the effects of shear, individually, on cells or blood vessels.
Seliktar et al. (U.S. Pat. No. 5,928,945) describes a bioreactor for producing cartilage in vitro, comprising a growth chamber, a substrate on which chondrocyte cells or chondrocyte stem cells are attached, and means for applying relative movement between a liquid culture medium and the substrate to provide a shear flow stress to the cells attached to the substrate.
In U.S. Pat. No. 5,899,937 Goldstein et al. describe a closed, sterile pulsatile loop for studying tissue valves. The system provides a tool to examine heart valve leaflet fibroblast function and differentiation as these are affected by mechanical loading, as well as an apparatus to provide heart valves seeded with suitable cells. The sterile pulsatile flow system which exposes viable tissue valves to a dynamic flow environment imitating that of the aortic valve.
Wolf et al. (U.S. Pat. No. 5,271,898) discloses an apparatus for testing blood/biomaterials/device interactions and characteristics, comprising a stepper-motor driven circular disc upon which a test vehicle is mounted. The test vehicle comprises a circular, closed loop of polymer tubing containing a check valve, and contains either the test materials, coating, or device. The apparatus generates pulsatile movement of the test vehicle. Oscillation of the test vehicle results in the pulsatile movement of fluid over its surface.
In U.S. Pat No. 6,205,871 B1, Saloner et al. disclose a panel of anatomically accurate vascular phantoms comprising a range of stenotic conditions varying from normal to critically stenosed (0% area reduction to greater than 99% reduction), and which phantoms are subjected to pulsatile flow of a blood mimic fluid.
Vilendrer (U.S. Pat. No. 5,670,708) discloses a device for measuring compliance conditions of a prosthesis under simulated physiologic loading conditions. The prosthesis includes stents, grafts and stent-grafts, which is positioned within a fluid conduit of the apparatus, wherein the fluid conduit is filled with a saline solution or other fluid approximating the physiological condition to be tested. The fluids are forced through the fluid conduit from both ends of the conduit in a pulsating fashion at a high frequency simulating systolic and diastolic pressures.
In U.S. Pat. No. 4,839,280 Banes describes an apparatus for applying stress to cell cultures, comprising at least one cell culture plate having one or more wells thereon, with each of the wells having a substantially planar base formed at least partially of an elastomeric membrane made of biocompatible polyorganosioxane composition, with the elastomeric membrane having an upper surface treated to permit cell growth and attachment thereto by means of the incorporation at the upper surface of a substance selected from the group consisting of an amine, a carboxylic acid, or elemental carbon, and vacuum means for controlling the elastomeric membrane to the pulling force of a vacuum. Banes (U.S. Pat. No. 6,218,178 B1) discloses an improvement, in the form of a loading station assembly for allowing stretching of a flexible cell culture membrane, the assembly comprising a planar member and a post extending from a surface of the planar member, an upper surface of the post being configured to support a flexible cell culture membrane, the planar member defining a passageway configured to allow fluid to flow through from one side of the planar member to an opposite side of the planar member, and wherein the flexible cell culture member is stretchable at a periphery of the upper surface towards the planar member.
In U.S. Pat. Nos. 4,940,853 and 5,153,136 Vanderburgh describes a method and apparatus for growing tissue culture specimens in vitro, respectively. The apparatus comprises an expandable membrane for receiving a tissue specimen thereon, a mechanism for expanding the membrane and the tissue specimen, and a controller for controlling the expanding mechanism. The controller is operative for applying an activity pattern to the membrane and a tissue specimen thereon which includes simultaneous continuous stretch activity and repetitive stretch and release activity. The continuous stretch and release activity simulate the types of activity to which cells are exposed in vivo due to growth and movement, respectively, and they cause the cells of tissue specimens grown in the apparatus to develop as three-dimensional structures similar to those grown in vivo.
In U.S. Pat Nos. 5,217,899 and 5,348,879 Shapiro et al. describe an apparatus and method for stretching cells in vitro, respectively. The inventions impart to a living culture of cells biaxial mechanical forces which approximate the mechanical forces to which cells are subjected in vivo. The apparatus includes a displacement applicator which may be actuated to contact and stretch a membrane having a living cell culture mounted thereon. Stretching of the membrane imparts biaxial mechanical forces to the cells. These forces may be uniformly applied to the cells, or they may be selectively non-uniformly applied.
Lee et al. (U.S. Pat No. 6,057,150) discloses a biaxial strain system for cultured cells that includes a support with an opening over which an elastic membrane is secured, a moveable cylinder coaxial with the opening and fitting closely but movably within the opening, and an actuating member that stabilizes and controls the position of the cylinder relative to the opening. The actuating member is coupled to the support by a threaded connection while engaging the movable cylinder. The degree of membrane stretch is accurately controlled by the rotation of the actuating member.
In U.S. Pat. No. 4,851,354 Winston et al. disclose an apparatus for mechanically stimulating cells, comprising an airtight well having an optically transparent compliant base of a biologically compatible material on which the cells may be grown and an optically transparent, removable cap, coupled with a ported, airtight reservoir which reservoir has an optically transparent base and which reservoir can be filled with pressuring media to create cyclic variations in hydrostatic pressure beneath the complaint base, causing the compliant base to deform and thereby exert a substantially uniform biaxial force on the cells attached thereto.
Lintilhac et al. (U.S. Pat. No. 5,406,853) disclose an instrument for the application of controlled mechanical loads to tissues in sterile culture. A slider which contacts the test subject is in force transmitting relation to a forcing frame. Tension, compressive and bending forces can be applied to the test subject, and force applied to the test subject is measured and controlled. A dimensional characteristic of the test subject, such as growth, is measured by a linear variable differential transformer. The growth measurement data can be used to control the force applied. Substantially biaxial stretching is achieved by placing the test subject on an elastic membrane stretched by an arrangement of members securing the elastic member to the forcing frame.
In U.S. Pat. No. 6,107,081 Feeback et al. disclose a unidirectional cell stretching device capable of mimicking linear tissue loading profiles, comprising a tissue culture vessel, an actuator assembly having a relatively fixed structure and an axially transformable ram within the vessel, at least one elastic strip which is coated with an extracellular matrix, and a driving means for axially translating the ram relative to the relatively fixed structure, and for axially translating the end portion of the elastic strap affixed to the ran relative to another, opposite end portion, for longitudinally stretching the elastic strap.
Nguyen et al. (U.S. Pat. No. 5,272,909) disclose a method and device for testing venous valves in vitro. The device comprises (a) a fixture for mounting a sample valve on a liquid flow path, (b) a muscle pump component and/or (c) respiratory pump component and/or (d) capacitance reservoir component and/or (e) vertical hydrostatic column component, all of the components being fluidly connected to the flow path to mimic the muscle pump, respiratory pump, capacitance and hydrostatic impedance effects of actual in situ venous circulation in the mammalian body. The muscle pump is designed to mimic effects caused by movement of the visceral organs and somatic muscles on a vein, while the respiratory pump is designed mimic the effects of normal cyclic variations in the intra-thoracic pressure due to the movement of the thoracic muscles and diaphragm. The combination of pumps of the present invention provides a means to examine the effects of pulsatile pressure, wall shear, stress, and circumferential strain, separately or in combination, on blood vessels or mammalian cells in vitro.
In U.S. Pat. No. 5,537,335 Antaki et al. disclose a fluid delivery apparatus in which a predetermined pressure waveform is introduced into a conduit, such as a human saphenous vein. By such exposure, the vein can be “arterialized”, meaning that it can be conditioned in preparation for its use in bypass surgery. An excised vein according to the inventors. The combination of pumps and the manner of controlling the degree of their being in phase or out-of-phase with each other provides a means to examine not only the effects of a blood pressure waveform, but also the effects of pulsatile pressure, wall shear stress, and circumferential strain, separately or in combination, on blood vessels or mammalian cells in vitro.
The most common WSS simulating systems utilize a 2-dimensional stiff surface, such as a glass slide, for the endothelial cell culture forming the wall of a parallel plate flow chamber. The WSS in these devices is usually steady because of difficulties in simulating pulsatile flow. Cyclic straining devices provide only strain, by stretching cells on a compliant membrane without flow. Both types of systems are thus limited by their design. However, no studies have been performed studying both parameters (WSS and CS) using cells grown on a single type of support surface because such a system, until now, has remained technologically unfeasible. The present invention addresses and solves this long-felt need by providing a system in which endothelial cells can be grown on a single support surface, and subjected to studies in which both wall shear stress and circumferential strain can be examined independently of each other.
The use of a silicone tube coated with endothelial cells was recently introduced, and provided the potential for simultaneous coupled pulsatile strain and shear stress. However, these tubes were used in flow simulators coupling pressure and flow that could only achieve phase angles (SPAs) of about 90-100 degrees; such a phase angle was inadequate for simulating coronary arteries, the most disease prone vessels in the circulation, because coronary arteries are characterized by a high SPA, on the order of approximately 250 degrees. These flow simulators were difficult to use and to produce replicable reliable results. The present invention overcomes this problem, by providing time-varying uniform cyclic pressure (and consequently CS) and pulsatile flow (and consequently WSS) in a 3-dimensional configuration over a complete range of SPAs, as a most complete physiologic environment.